The expression of the myosin heavy chain (MHC) multigene family in myogenic and non-myogenic tissues offers an excellent model system in which to study the controlling mechanisms of contractile protein biosynthesis. This information, in turn, will provide valuable information on general mechanisms of gene regulation in higher organisms. The major objective of this proposal is to use several approaches to examine the molecular mechanisms involved in the selective expression of the MHC genes in different cell types and stages of development. Fist, the complete nucleotide sequence of one embryonic gene will be analyzed and theprecise composition and structure of other members of the MHC multigene family will be determined by restriction mapping, R-loop and heteroduplex analysis, and DNA sequencing. The chromosomal location of the MHC genes in the mouse and human will also be determined. Secondly, the regulation of one embryonic gene expressed in the L6E9 myogenic cell line will be analyzed in wild type and a mutant cell line which is temperature sensitive (ts) for commitment. In this cell line the differentiated phenotype is reversible. The correlation of DNAse I sensitivity and cy tosine methylation with the transcription of this gene will be determined. Thirdly, the putative regulatory sequences of the MHC genes will be studied in in vitro cell-free assays as well as in vivo assays by introducing these sequences into competent mouse myogenic cells. The phenotype of modified regulatory sequences will also be determined using similar approaches. It is hoped that this combined approach, cellular and molecular, to study the different members of the MHC multigene family will uncover interesting new information on gene regulation in general and myogenesis in particular.